CN111110864A - Organic conjugated small molecule nano particle and preparation method and application thereof - Google Patents
Organic conjugated small molecule nano particle and preparation method and application thereof Download PDFInfo
- Publication number
- CN111110864A CN111110864A CN202010076208.7A CN202010076208A CN111110864A CN 111110864 A CN111110864 A CN 111110864A CN 202010076208 A CN202010076208 A CN 202010076208A CN 111110864 A CN111110864 A CN 111110864A
- Authority
- CN
- China
- Prior art keywords
- organic conjugated
- small molecule
- formula
- organic
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nanotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides an organic conjugated micromolecule nano particle and a preparation method and application thereof, wherein the nano particle comprises organic conjugated micromolecules; and polystyrene maleic anhydride for surface modification of the organic conjugated small molecule; the organic conjugated micromolecules are selected from one or more of structures shown in formula I, formula II, formula III and formula IV; the polystyrene maleic anhydride has the structure of formula V. The organic conjugated micromolecule in the organic conjugated micromolecule nano particle is used as a photosensitizer, and the amphiphilic molecule PSMA is used for surface modification, so that the water solubility of the nano particle is enhanced, the interaction with bacteria is facilitated, and meanwhile, the organic conjugated micromolecule nano particle has higher active oxygen generation efficiency, which shows that the organic conjugated micromolecule nano particle has phototoxicity; the cyano group attached indicates that it contributes significantly to bactericidal activity, indicating that it has dark toxicity. Therefore, the nano-particles have the synergistic effect of phototoxicity and dark toxicity, and can be used as an antibacterial drug for treating infection caused by bacteria. The method is simple, does not need any functional modification, has high synthesis yield and is easy to realize industrialization.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to organic conjugated micromolecule nanoparticles as well as a preparation method and application thereof.
Background
Bacterial infections threaten the life safety of millions of people each year, deprive more than 1700 million people of life, and are increasingly becoming a global public health problem. The traditional treatment methods include antibiotics, metal ions, quaternary ammonium ions and the like, but the methods are limited in application due to high cost, high toxicity, high harm to the environment and the like, and especially the drug resistance of bacteria to antibiotics causes the generation of multi-drug resistant bacteria, especially the multi-drug resistant gram negative bacteria. In this context, photodynamic antibacterial therapy (PDAT) has received widespread attention because of its non-invasive, selective, and high spatial resolution properties. In PDT, the light source, the photosensitizer and the oxygen molecule are three important components. Under the condition of illumination, the photosensitizer is activated to a singlet excited state, and the photosensitizer crosses to a triplet excited state through intersystem crossing, so that the energy of the triplet excited state is transferred to surrounding oxygen molecules when the triplet excited state returns to a ground state, and Reactive Oxygen Species (ROS) are generated, thereby achieving the antibacterial effect. Researchers want to obtain photosensitizers with strong light absorption energy, good light stability and good water solubility, but conventional photosensitizers such as organic dyes and phosphorescent transition metal complexes have difficulty meeting the above requirements. Compared with other photosensitizer materials, conjugated molecules are considered to be an ideal photosensitizer material due to the characteristics of special molecular structure, optical characteristics and low cost.
However, the existing hydrophobic conjugated molecules are difficult to interact with bacteria, so that the sterilization effect of the hydrophobic conjugated molecules is poor.
Disclosure of Invention
In view of the above, the present invention provides an organic conjugated small molecule nanoparticle, and a preparation method and an application thereof, wherein the organic conjugated small molecule nanoparticle has a synergistic bactericidal effect of phototoxicity and dark toxicity.
The invention provides an organic conjugated micromolecule nanoparticle, which comprises organic conjugated micromolecules;
and polystyrene maleic anhydride for surface modification of the organic conjugated small molecule;
the organic conjugated micromolecules are selected from one or more of structures shown in formula I, formula II, formula III and formula IV;
the polystyrene maleic anhydride has the structure of formula v:
preferably, the average particle diameter of the organic conjugated small molecule nanoparticles is 80-140 nm.
Preferably, the mass ratio of the organic conjugated micromolecule substance to the polystyrene maleic anhydride is (0.8-1.2) mol, (1.8-2.4) mg.
The invention provides a preparation method of organic conjugated micromolecule nanoparticles, which comprises the following steps:
mixing the organic conjugated micromolecule solution and the polystyrene maleic anhydride solution in a water phase to obtain a mixed solution;
and removing the organic solvent in the mixed solution, heating, concentrating and filtering to obtain the organic conjugated micromolecule nanoparticles.
Preferably, the solvent in the organic conjugated small molecule solution and the solvent in the polystyrene maleic anhydride solution are both selected from tetrahydrofuran.
Preferably, the concentration of the organic conjugated micromolecule solution is (0.8-1.2) mmol/L;
the concentration of the polystyrene maleic anhydride solution is (1.8-2.4) mg/mL.
Preferably, the temperature for increasing the temperature and concentrating is 90-99 ℃.
The invention provides an application of the organic conjugated micromolecule nanoparticles in the technical scheme or the organic conjugated micromolecule nanoparticles prepared by the preparation method in the technical scheme in the preparation of antibacterial drugs.
Preferably, the use thereof for the preparation of a medicament against gram-negative bacteria.
The invention provides an organic conjugated micromolecule nanoparticle, which comprises organic conjugated micromolecules; and polystyrene maleic anhydride (PSMA) surface-modified to the organic conjugated small molecule; the organic conjugated micromolecules are selected from one or more of structures shown in formula I, formula II, formula III and formula IV; the polystyrene maleic anhydride has a structure of formula V. The organic conjugated micromolecule in the organic conjugated micromolecule nano particle provided by the invention is used as a photosensitizer, and the amphiphilic molecule PSMA is used for surface modification, so that the water solubility of the nano particle is enhanced, the interaction with bacteria is facilitated, and meanwhile, the organic conjugated micromolecule nano particle has higher active oxygen generation efficiency, which shows that the organic conjugated micromolecule nano particle has phototoxicity; the attached cyano group indicates that it has an important contribution to bactericidal activity, indicating that it has dark toxicity. Therefore, the organic conjugated micromolecule nanoparticles provided by the application have the synergistic effect of phototoxicity and dark toxicity, and can be used as antibacterial drugs for treating infection caused by bacteria. In addition, the preparation method provided by the invention is simple, does not need any functional modification, has high synthesis yield and is easy to realize industrialization. The experimental results show that: the antibacterial effect of the organic conjugated micromolecule nano particles under the illumination condition reaches 99 percent.
Drawings
FIG. 1 shows the UV-VIS absorption spectrum and fluorescence emission spectrum of NPs 1-4;
FIG. 2 is a graph showing a particle size distribution of NPs 1-4;
FIG. 3 is a graph showing the active oxygen measurement curves of NPs 1-4;
FIG. 4 is Zeta potential characterization after NPs 1-4 of different concentrations reacted with E.coli;
fig. 5 a is an isothermal titration calorimetry curve after NPs1 reacts with e.coli; fig. 5 b is the isothermal titration calorimetry curve after NPs2 reacts with e.coli;
in FIG. 6, a is a graph of the sterilization efficiency of NPs 1-4 with different concentrations under the condition of keeping out of the sun; in FIG. 6, b is a graph of the sterilization efficiency of NPs 1-4 with different concentrations under exposure conditions;
FIG. 7 is a plate diagram of LB agar after NPs 1-4 (4.8. mu. mol/L) have reacted with E.coli;
FIG. 8 is a scanning electron microscope image of NPs 1-4 (4.8. mu. mol/L) after interaction with E.coli.
Detailed Description
The invention provides an organic conjugated micromolecule nanoparticle, which comprises organic conjugated micromolecules;
and polystyrene maleic anhydride for surface modification of the organic conjugated small molecule;
the organic conjugated micromolecules are selected from one or more of structures shown in formula I, formula II, formula III and formula IV;
the polystyrene maleic anhydride has the structure of formula v:
in the invention, the average particle diameter of the organic conjugated micromolecule nanoparticles is preferably 80-140 nm, and more preferably 100-110 nm. In a specific embodiment, the average particle diameter of the organic conjugated small molecule nanoparticles is 104.3 ± 2.2 nm.
The molecular weight of the polystyrene maleic anhydride is 1300-1700 g/mol, preferably 1400-1500 g/mol; in the specific embodiment of the invention, the molecular weight of the polystyrene maleic anhydride is 1500g/mol, and the polystyrene maleic anhydride is purchased from Tianjin Xiansi Biotechnology Co., Ltd. In the invention, the mass ratio of the amount of the organic conjugated micromolecule substance to the polystyrene maleic anhydride is preferably (0.8-1.2) mol, (1.8-2.4) mg, more preferably (0.9-1.1) mol, (1.9-2.4) mg; in a specific embodiment, the mass ratio of the organic conjugated micromolecule substance to the polystyrene maleic anhydride is 1.0mol:2.0 mg.
In the present invention, the organic conjugated small molecules with the structures of formula I, formula II, formula III and formula IV are all commercially available or prepared by methods well known to those skilled in the art. The organic conjugated small molecule of the structure I is preferably synthesized, characterized and characterized By electron donor-acceptors based on 9,9-diarylfluorene By Ouyang, Miet al From Wuuli HuaxueXuebao,27(6), 1516-; 2011 by the method described above; the organic conjugated small molecule with the structure of formula II is preferably selected from An aggregation-induced emission as a quantitative fluorescent sensor for cyanamide in aqueous medium By Fu, Guang-Liang; zhao, Cui-Hua FromTetrahedron (2013),69(6), 1700-1704); the organic conjugated small molecules of the structures of formula III and formula IV are preferably prepared according to the following general formula III-state empirical para-tertiary phenyl groups laterally with a diphenylamino group By ZHao, Cui-Hua; zhao, Yi-Hong; pan, Hong; fu, Guang-Liang From Chemical Communications (2011),47(19), 5518-5520.
The invention provides a preparation method of organic conjugated micromolecule nanoparticles, which comprises the following steps:
mixing the organic conjugated micromolecule solution and the polystyrene maleic anhydride solution in a water phase to obtain a mixed solution;
and removing the organic solvent in the mixed solution, heating, concentrating and filtering to obtain the organic conjugated micromolecule nanoparticles.
The method mixes the organic conjugated micromolecule solution and the polystyrene maleic anhydride solution in the water phase to obtain mixed solution. In the present invention, the solvent in the organic conjugated small molecule solution is preferably selected from tetrahydrofuran. The concentration of the organic conjugated micromolecule solution is preferably (0.8-1.2) mmol/L, and more preferably (0.9-1.1) mmol/L; in a specific embodiment, the concentration of the organic conjugated small molecule solution is 1 mmol/L. The solvent in the polystyrene maleic anhydride solution is preferably selected from tetrahydrofuran; the concentration of the polystyrene maleic anhydride solution is preferably (1.8-2.4) mg/mL, and more preferably (1.9-2.4) mg/mL; in a specific embodiment, the concentration of the polystyrene maleic anhydride solution is 2 mg/mL.
The organic conjugated micromolecule solution and the polystyrene maleic anhydride solution are preferably filtered by adopting 0.22 mu m organic phase filter membranes respectively for later use. The preparation method comprises the steps of adding the organic conjugated micromolecule solution and the polystyrene maleic anhydride solution into tetrahydrofuran, mixing uniformly, and pouring into water; the volume ratio of the organic conjugated micromolecule solution to the polystyrene maleic anhydride solution to the tetrahydrofuran to the water is preferably 0.18-0.22: 3.5-4.5: 9-11, more preferably 0.19-0.21: 3.8-4.2: 9.5-10.5, and most preferably 0.2:4: 10. In the specific embodiment, 200 mul of organic conjugated micromolecule solution and 200 mul of polystyrene maleic anhydride solution are mixed into 4mL of tetrahydrofuran, and then poured into 10mL of water. The organic conjugated micromolecule solution and the polystyrene maleic anhydride solution are respectively taken and added into tetrahydrofuran, and preferably, the mixture is ultrasonically mixed. And pouring the mixture into water, and continuing performing ultrasonic treatment for 4-6 min.
After a mixed solution is obtained, the organic solvent in the mixed solution is removed, heated, concentrated and filtered to obtain the organic conjugated micromolecule nano particles. The method preferably adopts a nitrogen introducing mode to remove the organic solvent in the mixed solution; in a specific embodiment, the nitrogen is introduced for 55-65 min. The temperature for heating and concentrating is preferably 90-99 ℃, and more preferably 96-99 ℃; the concentration is preferably carried out in a water bath, and in a specific embodiment, the temperature of the water bath concentration is 99 ℃. The method is preferably concentrated to 45-60% of the volume of the mixed solution; in a specific example, the water bath was concentrated to 5 mL.
After concentration, the concentrated product is filtered again to obtain the organic conjugated micromolecule nano particles. The invention preferably employs a 0.22 μm aqueous phase filtration membrane for filtration.
The invention provides an application of the organic conjugated micromolecule nano particle in the technical scheme in preparation of antibacterial drugs. The invention preferably applies the antibacterial agent to the preparation of the anti-gram-negative bacteria medicament. In a specific embodiment, the gram-negative bacterium is escherichia coli.
In a specific embodiment, the OD of the bacterial liquid6001.0. The method adopts 1.6-4.8 mu mol/L nano particles to mix with the bacterial liquid; specifically, the concentration was 1.6. mu. mol/L, 3.2. mu. mol/L, and 4.8. mu. mol/L, respectively.
The organic conjugated micromolecule nano particle provided by the invention is applied to antibiosis, can be used for preparing antibacterial drugs, expands antibacterial materials and realizes effective photodynamic killing on pathogenic bacteria.
After the organic conjugated micromolecules are modified by PSMA, the water solubility is enhanced, and the interaction between the organic conjugated micromolecules and bacteria is facilitated. Under the irradiation of white light, electrons of the organic conjugated small molecule nano particles are transferred from a ground state to an excited state after receiving energy, the electrons are transferred to a triplet state through an intersystem crossing mode, and when the triplet state returns to the ground state, surrounding oxygen molecules are sensitized to generate active oxygen species. When active oxygen free radicals exist in the system, the active oxygen free radicals and esters on cell membranes can generate peroxidation, so that the cell membranes of bacteria are cracked, cytoplasm flows outwards, and effective photodynamic killing on the bacteria is realized. Meanwhile, the cyano group connected with the organic conjugated micromolecule is found to have an important contribution effect on the bactericidal activity and certain dark toxicity, and the prepared nano particle has the synergistic bactericidal effect of phototoxicity and dark toxicity.
In order to further illustrate the present invention, the following examples are provided to describe in detail the organic conjugated small molecule nanoparticles and the preparation method and application thereof, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of organic conjugated small molecule nano particle by nano precipitation method
The method comprises the following specific steps: dissolving the organic conjugated small molecules with the structures of formula I, formula II, formula III and formula IV in Tetrahydrofuran (THF) solution respectively to prepare the concentration of 1mmol/L, and filtering with organic phase filter membrane (0.22 μm) for standby. PSMA was dissolved in THF solution at a concentration of 2mg/mL, and filtered through an organic phase filter (0.22 μm) for further use. And (3) adding 200 mu L of each of the two solutions into a THF solution containing 4mL of the solution, and uniformly mixing the solution by ultrasonic waves. Then poured into a flask containing 10mL of ultrapure water and sonicated for about 5 min. And introducing nitrogen for about 1h, removing THF in the system, continuously introducing nitrogen, performing water bath concentration at 99 ℃ in a water bath kettle until the system is 5mL, and filtering with an aqueous phase filter membrane (0.22 mu m) to obtain organic conjugated small molecule nanoparticle solutions which are respectively marked as NPs1, NPs2, NPs3 and NPs 4.
FIG. 1 is the UV-visible absorption spectrum and fluorescence emission spectrum of the nanoparticles prepared above, and FIG. 2 is the particle size distribution diagram of the nanoparticles prepared above, and the average particle size can reach 104.3 + -2.2 nm.
Example 2
Active oxygen test of prepared organic conjugated small molecule nano particle
Activated 2, 7-Dichlorofluorescein (DCFH)
4.8mg of 2, 7-dichlorofluorescein diacetate (DCFH-DA) was placed in a 1.5mL centrifuge tube, 1mL of ethanol was added, and the mixture was mixed to obtain a 10mmol/L stock solution of DCFH-DA. 50 mu L of stock solution is taken and put into a 1.5mL centrifuge tube, 450 mu L of ethanol is added and mixed evenly, and the mixture is diluted to 1 mmol/L. Adding 2ml of 0.01M NaOH solution, mixing uniformly, and activating at 25 ℃ for 30 min. Adding 10ml Phosphate Buffer Solution (PBS) with pH value of 7.4 into the activated liquid, and storing in an ice box in a dark place for later use.
The control group was DCFH alone, 1mL of activated DCFH solution plus 100. mu.LPBS, illuminated directly, measured every 1min of illumination, for a total time of 4 min.
And (3) ROS measurement:
and (3) putting 1mL of activated liquid into a cuvette, adding 100 mu L of NPs solution, uniformly mixing, and measuring the fluorescence emission spectrum of the mixed solution by using a fluorescence spectrometer. Then irradiating for 1min (white light source intensity is 0.5 mW/cm)2) Measuring the fluorescence spectrum once, and repeating the steps. The total time of measurement was 4 min. The fluorescence spectrum excitation wavelength is 488nm, the collection wavelength range is 500-700nm, and the fluorescence intensity data at 524nm is taken as a graph.
In order to test the efficiency of the prepared organic conjugated small molecule nano-particles NPs 1-4 for generating active oxygen, an active oxygen test is carried out, and the result is shown in FIG. 4. When NPs1 and NPs2 are present in the system, the intensity is (0.5 mW/cm) under the illumination condition2) The fluorescence intensity of DCFH is obviously enhanced along with the increase of illumination time, which indicates that NPs1 and NPs2 have higher active oxygen generation efficiency and the possibility of photodynamic sterilization exists. Compared with the control group, the fluorescence intensity of the DCFH (dichloro-diphenyl-trichloroethane) of the NPs3 and the NPs4 is increased slowly, which indicates that the sterilization efficiency of the NPs3 and the NPs4 is not mainly phototoxicity, and indicates that the subsequent sterilization efficiency is related to the molecular structure and the connected cyano group.
Example 3
Measuring potential change and Isothermal Titration Calorimetry (ITC) after interaction of coli and organic conjugated micromolecule nanoparticles
In order to further explore the action mechanism of the organic conjugated small molecule nanoparticles and E.coli, the potential measurement and ITC measurement are carried out, and the experimental steps are as follows:
potentiometric determination
Take E.coli (OD)6001.0), and (0 μmol/L, 1.6 μmol/L, 3.2 μmol/L, and 4) were added to the suspension at different concentrations.8. mu. mol/L) NPs, and finally adding Phosphate Buffered Saline (PBS) to make the final volume 500. mu.L, mixing well, and incubating at 37 ℃ for 30 min. After centrifugation (7100rpm, 2min), unbound NPs were removed. The strain was resuspended in 1mL of ultrapure water, stored in an ice bath and ready to measure potential.
The results of the experimental data in FIG. 4 show that the surface potential of E.coli has no obvious change after the E.coli is mixed and cultured with NPs (0. mu. mol/L, 1.6. mu. mol/L, 3.2. mu. mol/L and 4.8. mu. mol/L) with different concentrations. The potential change trend is probably caused by the fact that the prepared NPs have small particle sizes and do not interact with negatively charged groups on the cell membranes on the surfaces of the bacteria after being attached to the E.coli surfaces, and therefore the potential is not obviously changed.
ITC assay
Subsequently, NPs1 and NPs2 were selected as representatives for the ITC curve determination. Adding PBS solution and E.coli bacteria solution into a sample pool respectively, adding NPs1 and NPs2 solution into the sample pool by injection, and stirring by a stirrer at the rotating speed of 60 rpm. And measuring the temperature difference between the measured temperature and the blank group by a temperature sensing device of the instrument, and fitting an ITC curve to obtain corresponding thermodynamic parameters. The experimental conditions are 25.00 +/-0.01 ℃. The experiment was repeated so that the standard deviation was within ± 4%.
As shown in fig. 5, when NPs1 is added to the bacterial liquid, an exothermic reaction occurs after NPs1 is bonded to the surface of the bacteria, the amount of heat released by the system gradually decreases with the increase of the addition amount of the NPs1 solution, and the final trend is close to 0, which indicates that the bonding between NPs1 and e.coli is close to a saturation state, the ITC curve approximately shows an S-shape, and a similar variation trend can also be observed in NPs 2. Through the variation trend of the curve, the corresponding thermodynamic parameters are obtained. Binding constants K of NPs1 and NPs2 to e.coli are large (2.45 × 10, respectively)7±7.45×106，1.79×107±2.51×106) Indicating that NPs1 and NPs2 have strong binding force with E. The surface potentials of the NPs1 and the NPs2 nanoparticles are negative, so that the electrostatic interaction with bacteria is eliminated. According to the changes of thermodynamic parameters delta H and delta S of NPs1 and NPs2, the reason that the bonding force of the two NPs and the bacteria is strong is analyzed to be the interaction result of the hydrophobic structures on the surfaces of the NPs and the hydrophobic groups on the surfaces of the bacteria.And meanwhile, carboxyl groups on the surface of the nano particles can possibly have hydrogen bond interaction with proteins and amino acids on the surface of the bacteria, which shows that the NPs 1-2 and E.
Example 4 antibacterial experiment of organic conjugated small molecule nanoparticles
100 mu L of E.coli bacteria were mixed with NPs (1.6. mu. mol/L, 3.2. mu. mol/L, 4.8. mu. mol/L) at 37 ℃ and cultured for 30 min. Then irradiating for 30min under white light source with intensity of 75mW/cm2. Coli serially diluted 1 × 10 in PBS solution after the end of light irradiation4And (4) doubling. mu.L of the bacterial suspension was applied to an agar medium containing ampicillin (100mg/mL), and cultured at 37 ℃ for 12 hours. The control group was protected from light for 30min during the light step. The diameter of the selected disposable sterilized culture dish is 90 mm. After the culture was completed, plate counting was performed, and the bacteriostatic ratio (IR) was calculated according to the following formula:
c is the total number of the bacterial colonies in the experimental group in the sterilization experiment, C0The total number of colonies in the blank group was not added with NPs in the sterilization experiment
The antibacterial result is shown in fig. 6, after NPs1 and E.coli act, the antibacterial rate is increased from 14% to 28% along with the increase of the concentration (1.6-4.8 mu mol/L) of NPs1 under the condition of keeping out of the sun, and the bactericidal effect is not obvious. Under the condition of illumination, the sterilization efficiency is obviously improved, the sterilization effect reaches 62% under the concentration of 4.8 mu mol/L, the phototoxicity is proved to be superior to dark toxicity, and the fact that NPs1 is mainly sterilized in a mode of generating ROS after illumination is shown. The dark toxicity of the NPs is gradually enhanced along with the increase of the concentration of the NPs2, and the sterilization effect reaches 89% at the final concentration of 4.8 mu mol/L. Under the condition of illumination, the phototoxicity of the bactericidal composition is also superior to the dark toxicity of the bactericidal composition, and the bactericidal effect reaches 99% at the final concentration, which indicates that the NPs2 has the synergistic effect of phototoxicity and dark toxicity. The NPs3 is slightly improved under the condition of illumination in the same concentration range than under the condition of keeping out of the sun, and is related to the lower efficiency of generating ROS under the condition of illumination, possibly due to the molecular structure of the nano-particle. The bactericidal effect of NPs4 is shown in the purple histogram, and the bactericidal effect does not change much under the conditions of dark and light, and the result suggests that NPs4 is mainly sterilized in a dark toxic manner. NPs2, NPs3 and NPs4 have the same carbon skeleton structure, but are different in connected conjugated groups, so that comparative analysis can be carried out, and the sterilization experiment result shows that the cyano group has important contribution to the sterilization activity.
At a high concentration (4.8. mu. mol/L), colonies grew in LB medium as shown in FIG. 7, and it was found that the number of colonies was significantly decreased compared to the control group after the addition of nanoparticles. In order to further observe the bacterial morphology, the bacterial morphology was further observed at a high concentration (4.8. mu. mol/L) using a Scanning Electron Microscope (SEM), and the results are shown in FIG. 8. When NPs are not added in the system, the E.coli shows complete bacterial morphology under the conditions of light shielding and illumination, complete boundaries among bacteria can be observed, a good growth state is shown, and meanwhile, illumination has no obvious influence on the growth of the bacteria. Under the condition of high concentration, the cell membrane on the surface of the bacteria begins to deform, dent and the like under the condition of keeping out of the sun; under the condition of illumination, the surface of the bacteria is deepened, and phenomena such as cracking, fusion and the like occur, which are consistent with the results of antibacterial experiments.
In conclusion, the organic conjugated micromolecule nanoparticles are prepared by the method, and can be used for realizing effective photodynamic killing on E. The prepared nanoparticles NPs1 and NPs2 have high active oxygen generation efficiency under the irradiation of white light, and have the possibility of photodynamic sterilization. Through the measurement of potential and ITC, the non-specific binding of the organic conjugated small molecule nano particles to bacteria is shown. The sterilization experiment results show that NPs1 and NPs2 can realize effective photodynamic killing on E.coli and have certain dark toxicity. NPs2, NPs3 and NPs4 have similar carbon skeleton structures, and experimental results show that cyano groups have important contribution to sterilization of e.
From the above embodiments, the present invention provides an organic conjugated small molecule nanoparticle, which includes an organic conjugated small molecule; and polystyrene maleic anhydride (PSMA) surface-modified to the organic conjugated small molecule; the organic conjugated micromolecules are selected from one or more of structures shown in formula I, formula II, formula III and formula IV; the polystyrene maleic anhydride has a structure of formula V. The organic conjugated micromolecule in the organic conjugated micromolecule nano particle provided by the invention is used as a photosensitizer, and the amphiphilic molecule PSMA is used for surface modification, so that the water solubility of the nano particle is enhanced, the interaction with bacteria is facilitated, and meanwhile, the organic conjugated micromolecule nano particle has higher active oxygen generation efficiency, which shows that the organic conjugated micromolecule nano particle has phototoxicity; the attached cyano group indicates that it has an important contribution to bactericidal activity, indicating that it has dark toxicity. Therefore, the organic conjugated micromolecule nanoparticles provided by the application have the synergistic effect of phototoxicity and dark toxicity, and can be used as antibacterial drugs for treating infection caused by bacteria. In addition, the preparation method provided by the invention is simple, does not need any functional modification, has high synthesis yield and is easy to realize industrialization. The experimental results show that: the antibacterial effect of the organic conjugated micromolecule nano particles under the illumination condition reaches 99 percent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. An organic conjugated small molecule nanoparticle comprises an organic conjugated small molecule;
and polystyrene maleic anhydride for surface modification of the organic conjugated small molecule;
the organic conjugated micromolecules are selected from one or more of structures shown in formula I, formula II, formula III and formula IV;
the polystyrene maleic anhydride has the structure of formula v:
2. the organic conjugated small molecule nanoparticle according to claim 1, wherein the average particle diameter of the organic conjugated small molecule nanoparticle is 80-140 nm.
3. The organic conjugated micromolecule nanoparticles as claimed in claim 1, wherein the mass ratio of the substance of the organic conjugated micromolecules to the polystyrene maleic anhydride is (0.8-1.2) mol, (1.8-2.4) mg.
4. A method for preparing the organic conjugated small molecule nanoparticles of any one of claims 1 to 3, comprising the following steps:
mixing the organic conjugated micromolecule solution and the polystyrene maleic anhydride solution in a water phase to obtain a mixed solution;
and removing the organic solvent in the mixed solution, heating, concentrating and filtering to obtain the organic conjugated micromolecule nanoparticles.
5. The preparation method according to claim 4, wherein the solvent in the organic conjugated small molecule solution and the solvent in the polystyrene maleic anhydride solution are both selected from tetrahydrofuran.
6. The preparation method according to claim 4, wherein the concentration of the organic conjugated small molecule solution is (0.8-1.2) mmol/L;
the concentration of the polystyrene maleic anhydride solution is (1.8-2.4) mg/mL.
7. The method according to claim 4, wherein the temperature of the concentration at elevated temperature is 90 to 99 ℃.
8. Use of the organic conjugated small molecule nanoparticles according to any one of claims 1 to 3 or the organic conjugated small molecule nanoparticles prepared by the preparation method according to any one of claims 4 to 7 in preparation of antibacterial drugs.
9. Use according to claim 8, for the preparation of a medicament against gram-negative bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010076208.7A CN111110864B (en) | 2020-01-21 | 2020-01-21 | Organic conjugated small molecule nano particle and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010076208.7A CN111110864B (en) | 2020-01-21 | 2020-01-21 | Organic conjugated small molecule nano particle and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111110864A true CN111110864A (en) | 2020-05-08 |
| CN111110864B CN111110864B (en) | 2022-04-05 |
Family
ID=70491902
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010076208.7A Active CN111110864B (en) | 2020-01-21 | 2020-01-21 | Organic conjugated small molecule nano particle and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111110864B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113045455A (en) * | 2021-03-30 | 2021-06-29 | 武汉工程大学 | Aggregation-induced emission photosensitizer with near-infrared emission and high singlet oxygen yield and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030224525A1 (en) * | 2002-05-30 | 2003-12-04 | The University Of Chicago | Enhanced photophysics of conjugated polymers |
| CN109970630A (en) * | 2019-04-23 | 2019-07-05 | 四川大学 | A kind of two-photon fluorescence probe and its preparation method and application of energy targetted mitochondria |
-
2020
- 2020-01-21 CN CN202010076208.7A patent/CN111110864B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030224525A1 (en) * | 2002-05-30 | 2003-12-04 | The University Of Chicago | Enhanced photophysics of conjugated polymers |
| CN109970630A (en) * | 2019-04-23 | 2019-07-05 | 四川大学 | A kind of two-photon fluorescence probe and its preparation method and application of energy targetted mitochondria |
Non-Patent Citations (4)
| Title |
|---|
| GUANGHONG FU ET AL.: "An aggregation-induced emissive chromophore as a ratiometric fluorescent sensor for cyanide in aqueous media", 《TETRAHEDRON》 * |
| HUANXIANG YUAN: "Aggregation-induced emission nanoparticles with NIR and photosensitizing characteristics for resistant bacteria elimination and real-time tracking", 《MATERIALS CHEMISTRY FRONTIERS》 * |
| YUCHAO LIANG ET AL.: "3. Conjugated Polymer and Triphenylamine Derivative Codoped Nanoparticles for Photothermal and Photodynamic Antimicrobial Therapy", 《ACS APPL. BIO MATER.》 * |
| 袁焕祥: "双模式共轭聚合物纳米粒子的制备及其在病原菌杀伤方面的应用", 《第十一届全国化学生物学学术会议论文摘要）（第二卷）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113045455A (en) * | 2021-03-30 | 2021-06-29 | 武汉工程大学 | Aggregation-induced emission photosensitizer with near-infrared emission and high singlet oxygen yield and preparation method and application thereof |
| CN113045455B (en) * | 2021-03-30 | 2022-07-19 | 武汉工程大学 | Aggregation-induced emission photosensitizer with near-infrared emission and high singlet oxygen yield and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111110864B (en) | 2022-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022007153A1 (en) | Tumor-targeting nanodrug simultaneously enclosing photosensitizer and protein, and use thereof | |
| Wei et al. | A graphene oxide based smart drug delivery system for tumor mitochondria-targeting photodynamic therapy | |
| Xia et al. | An upconversion nanoparticle–Zinc phthalocyanine based nanophotosensitizer for photodynamic therapy | |
| Zhao et al. | Multifunctional core–shell upconverting nanoparticles for imaging and photodynamic therapy of liver cancer cells | |
| Wang et al. | Nanomaterials and singlet oxygen photosensitizers: potential applications in photodynamic therapy | |
| Guo et al. | Rose Bengal-decorated silica nanoparticles as photosensitizers for inactivation of gram-positive bacteria | |
| Kalluru et al. | Unprecedented “All‐in‐One” Lanthanide‐Doped Mesoporous Silica Frameworks for Fluorescence/MR Imaging and Combination of NIR Light Triggered Chemo‐Photodynamic Therapy of Tumors | |
| Zhang et al. | Synthesis of porphyrin-conjugated silica-coated Au nanorods for synergistic photothermal therapy and photodynamic therapy of tumor | |
| CN103566381A (en) | Multifunctional magnetic nano particle and preparation method thereof | |
| JP2012512817A (en) | Calcium phosphate nanoparticles used as a carrier system for photodynamic therapy | |
| EP3292409B1 (en) | Particle comprising at least one ferrimagnetic or ferromagnetic iron oxide nanoparticle associated with at least one compound for medical or cosmetic use | |
| Tan et al. | Magnetic iron oxide modified pyropheophorbide-a fluorescence nanoparticles as photosensitizers for photodynamic therapy against ovarian cancer (SKOV-3) cells | |
| Kotagiri et al. | Selective pathogen targeting and macrophage evading carbon nanotubes through dextran sulfate coating and PEGylation for photothermal theranostics | |
| Idowu et al. | Photophysicochemical behaviour and antimicrobial properties of monocarboxy Mg (II) and Al (III) phthalocyanine-magnetite conjugates | |
| Cheng et al. | Preparation, characterization and in vitro photodynamic therapy of a pyropheophorbide-a-conjugated Fe 3 O 4 multifunctional magnetofluorescence photosensitizer | |
| Ning et al. | Hydrothermal derived protoporphyrin IX nanoparticles for inactivation and imaging of bacteria strains | |
| CN106474474B (en) | Photo-thermal nano particle based on peptide and photosensitizer, preparation method and application thereof | |
| Yan et al. | Green synthesis of carbon quantum dots from plant turmeric holds promise as novel photosensitizer for in vitro photodynamic antimicrobial activity | |
| WO2022257855A1 (en) | H-bn/mos2 nanoprobe capable of achieving targeted photothermal and chemical synergistic therapy, preparation method therefor, and application thereof | |
| CN111803629A (en) | Organic-inorganic hybrid multifunctional biological material based on nano cellulose crystals and preparation method and application thereof | |
| Shabangu et al. | Susceptibility of Staphylococcus aureus to porphyrin-silver nanoparticle mediated photodynamic antimicrobial chemotherapy | |
| CN111110864B (en) | Organic conjugated small molecule nano particle and preparation method and application thereof | |
| Li et al. | Water-soluble porphyrin-based nanoparticles derived from electrostatic interaction for enhanced photodynamic therapy | |
| Bunin et al. | Phthalocyanine aggregates in the photodynamic therapy: dogmas, controversies, and future prospects | |
| Kulvelis et al. | Enhancement of singlet oxygen generation of radachlorin® conjugated with polyvinylpyrrolidone and nanodiamonds in aqueous media |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |